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First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02366

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Polarity of the ascidian egg cortex and relocalization of cER and mRNAs in the early embryo

François Prodon^1,*, Philippe Dru¹, Fabrice Roegiers² and Christian Sardet^1,

¹ BioMarCell, UMR7009 Biologie du Développement, CNRS/Université Pierre et Marie Curie, Station Zoologique, Observatoire, Villefranche sur Mer 06230, France
² Institute for Cancer Research, Fox Chase Cancer Center, 333 Cottman Avenue, R356, Philadelphia, PA 19111, USA

View larger version (165K): [in a new window]   Fig. 1. Polarity along the animal-vegetal (a-v) axis of the ascidian oocyte (see Movies 1-3 in supplementary material). (A1,A2) Confocal equatorial section (30 µm from surface) showing the distribution of the ER network (A1) and the 7-8 µm thick mitochondria-rich subcortical myoplasm (m) (A2). (A3,A4) Enlarged views of the ER-poor myoplasm. (B) Electron-microscopy section of the vegetal-pole region showing the ER network (red), ER microdomains (ER), cER near the plasma membrane (arrowheads), mitochondria (m, green) and yolk platelets (YP, blue). (C1,C2) Confocal subcortical section (3 µm from surface) passing through the myoplasm (m). (C3,C4) Enlarged views of the transition zone at the edge of the myoplasm (m). (D1,D2) High-magnification confocal cortical sections (1 µm from surface) of the cER network in the vegetal (D1) and animal hemispheres (D2). (E) ER around the meiotic spindle (ms, arrowhead) at the animal pole. (F1,F2) Equatorial (F1, 30 µm from the surface) and cortical (F2, 1 µm from surface) confocal sections of fluorescent in situ localization of Ci-PEM1 RNAs (arrowheads). (F3) Confocal cortical view (1 µm from surface) of the vegetal hemisphere at higher magnification. Ci-PEM1 RNA signal appears as a reticulated network. (a) Animal pole. (v) Vegetal pole. All confocal acquisitions are made on Ciona intestinalis oocytes, the electron micrograph is from a section of a Phallusia mammillata oocyte.

View larger version (84K): [in a new window]   Fig. 2. (I) Isolation of cortices from oocytes, zygotes and embryos. (IA). Eggs or synchronous embryos deposited into a drop of cortex buffer and allowed to attach to a polylysine-coated coverslip are sheared with a stream of cortex buffer from a Pasteur pipette. (IB) Fields of isolated cortices are labelled, mounted and observed using epifluorescence or confocal microscopy. (IC) The position of grey coverslips (c) indicate the origin of the cortical fragments isolated from oocytes, zygotes or embryos selected for observation. cER is red, the myoplasm is green, microtubules in light blue and DNA in dark blue. a, animal; v, vegetal; cp, contraction pole; d, dorsal; P, posterior; A, anterior. (ID) Field of cortices isolated from unfertilized eggs and labelled for cER. (II) Isolated cortices from oocytes retain a-v polarity. (IIA1) Cortex isolated from an oocyte labelled for cER (red) and microfilaments (yellow). Regions corresponding to the animal (a) and vegetal (v) hemispheres are shown. (IIA2) Detail of the region boxed in IIA1. (IIB1-B3) Cortex isolated from an oocyte. cER is double labelled with DiIC16(3) (B1, red) and by immunofluorescence for ribosomes (B2, green). Merged image of cER network and ribosomes (B3, colocalization in yellow). Dotted line indicates the edge of isolated cortex. Arrowhead indicates cytoplasmic ER that has fallen onto the coverslip during the shearing process (B3, arrowhead). (IIC1-D2) Fragments of cortices isolated from an oocyte labelled for cER (C1, red) and Ci-PEM1 (C2, green) (IID1,D2). Cortex isolated and labelled after KCl-puromycin treatment; labelled for cER (D1) and ribosomes (D2). (IIE). Replica of a fragment of an isolated oocyte cortex (fast-freezing deep-etching electron microscopy). Cortical ER is red, microfilaments orange and particles on the cER surface yellow. Most particles are the size of ribosomes (arrowheads), coated pits are blue and the internal side of the plasma membrane (PM) is grey. All cortices were obtained from Ciona oocytes except for E, which is from Phallusia.

View larger version (76K): [in a new window]   Fig. 3. Amplification of cortical polarity after fertilization (first major phase). (A1-C3) Zygotes and cortices during the contraction wave (see Movie 4 in supplementary material). (A1-A4) Confocal subcortical sections (3 µm from surface) of a zygote during the fertilization contraction wave, showing the distribution of cER network and mitochondria-rich subcortical domain (myoplasm). (B) Confocal equatorial section (35 µm from surface) of Ci-PEM1 RNA fluorescent in situ localization (arrowheads). Dotted line marks the edge of zygote. (C1) Cortex isolated from the vegetal-pole region during the contraction. Merged images of cER (red) and microfilaments (green) show colocalization in yellow. The external dotted line shows the edge of cortex and the internal dotted line shows the area of the highest cER accumulation. (C2,C3) Enlarged views of the region boxed in C1 showing cER (C2, red) accumulation (arrowhead) and corresponding microfilaments (C3, green). (D1-J1) Zygotes and cortices at the end of the contraction wave. (D1,D2) Confocal cortical to subcortical section (1 µm to 3-4 µm from the surface). (Insert) Merged image of cER (red) and mitochondria (green) in the vegetal/contraction pole region. (E) Cortical section of the vegetal/contraction pole and future dorsal pole of the embryo (cp/d) after a double labelling for cER (red) and mitochondria (green). (F1,F2) Equatorial (F1, 40 µm from surface) and surface (F2) sections of the vegetal/contraction pole region after labelling surface sugars with fluorescent ConA. (G) Equatorial section (35 µm from surface) of Ci-PEM1 RNA fluorescent in situ localization signal (arrowheads). The dotted line marks the edge of zygote. (H) Merged image of a cortex triple labelled for cER (red), microfilaments (green) and microtubules (blue). The yellow area indicates the vegetal/contraction pole (cp/d) area, which is rich in both cER and microfilaments. sa, sperm aster. (I1,I2) Detail of microtubules (MT) of a sperm aster (I2). This area corresponds to an accumulation of ER in the cortex (I1). (J1,J2) Detail of a cortex isolated from the vegetal/contraction pole double labelled for cER (J1) and surface sugars (J2). Whole-zygote confocal sections are from Ciona and cortices are from Phallusia zygotes. Dotted line indicates the border of the vegetal/contraction pole.

View larger version (103K): [in a new window]   Fig. 4. Cortical polarity along the anteroposterior axis (second major phase). (A1-A3) Successive confocal equatorial (40 µm from surface) sections of a zygote (31-41 minutes after fertilization) double labelled for ER (red) and mitochondria (green) (time-lapse confocal microscopy); see Movies 4 and 5 in supplementary material). The direction of posterior translocation of the myoplasm is indicated by the white arrow and that of the cER by the white arrowhead. DiI indicates the injected DiIC16(3) dye droplet. (B,C) Detection of the fluorescent in situ hybridization signal for CiPEM-1 RNA during (B) and at the end (C) of the second major phase of reorganization (arrowheads). (D1-D4) Posterior view of the CiPEM-1 RNA signal at the end of the second phase. Views at higher magnification of the CiPEM-1 RNA signal corresponding to the boxed area in (D1), 4 µm (D2; scale bar, 5 µm) and 1 µm (D3; scale bar, 5 µm) under the cell surface. (D4) A view at higher magnification of in situ localization of CiPEM-1 in the boxed area in D3 (scale bar, 2.5 µm). (E) Posterior view of astral microtubules. (F) Equatorial view of a zygote after labelling surface sugars with fluorescent ConA. Labelled sugars remain centred around future dorsal pole (d) region (arrowheads). (G) Microfilament labelling of surface (arrowheads). (H1-H3,I1-I3) Cortex isolated from a zygote (40 minutes after fertilization) along the posterior-dorsal (P-d) axis. The cortical fragment is double labelled for cER (H1,I1, red) and Ci-PEM1 (H2,I2, green). (I1-I3) Enlarged regions corresponding to the boxed areas in (H). (H3,I3,J3) Merged images in which colocalized cER and Ci-PEM1 are shown in yellow (I3). (J1-J3) Cortical fragment from the posterior cortex treated with KCl-puromycin and labelled for cER (J1, red) and Ci-PEM1 RNA (J2, green). (J3) Merged images shows that little Ci-PEM1 signal remains (arrowhead). (K1,K2) Fragment of isolated posterior cortex labelled for cER (K1, red) and microtubules (K2, white). a, animal; d, dorsal; P, posterior. All observations are made in Ciona except for F,G, which were made in Phallusia.

View larger version (112K): [in a new window]   Fig. 5. Cortical polarity of 8-16 cell stage embryos (first unequal cleavage). (A1-A3) Living embryo double labelled for ER (red) and mitochondria (green); time-lapse confocal microscopy of eight-cell (A1), 16-cell (A2) and 32-cell (A3) stages (see Movie 6 in supplementary material). Arrowheads indicate the location of the cER/mRNA-rich CAB. Inserts show full views of embryos at the eight-cell stage (lateral view) and at the 16- and 32-cell stages (vegetal views). (B,C) Fixed embryos double labelled for Ci-PEM1 RNA by fluorescent in situ hybridization (white) and DNA (blue). Arrowheads show the mRNA-rich CAB at the beginning (interphase) of the eight-cell stage (B) and at the end (mitosis) of the eight-cell stage (C). (D) High-magnification confocal cortical section (6 µm from surface) showing Ci-PEM1 RNA signal at the end of the eight-cell stage. (E1,E2) Electron-microscopy section across the CAB of an eight-cell-stage embryo. The dotted area encircles the CAB. Enlarged view of the CAB is shown in (E1). ER tubules (cER, arrowhead) are red, mitochondria green and yolk platelets (YP) blue. Notice the presence of electron-dense matrix. Scale bars, 5 µm (E1) and 1 µm (E2). (F) Confocal observation of cortical fragments isolated from the posterior vegetal region of an eight-cell-stage embryo and labelled for cER (red). (Insert, top right) Lower-magnification view of four cortical imprints from two vegetal posterior B4.1 and two animal posterior b4.2 blastomeres. Arrowheads indicate the cER accumulations that characterize the isolated moustache-shaped CAB. (Insert, bottom right) Higher-magnification view of cER accumulation observed in F. (G) Epifluorescence observation of the CAB isolated in a more condensed state and labelled for cER (arrowheads). Dotted lines show the edges of cortical fragments corresponding to blastomeres. (H1,H2) Confocal section of isolated cortex with condensed CAB double labelled for cER (H1) and microtubules (H2). Dotted lines delimit cortical fragments. (I) Confocal section of isolated cortex labelled for microfilaments in the area of a condensed CAB (arrowheads). (J) Isolated CAB double labelled for cER (red) and ribosomes (green). Merged images show colocalization of cER and ribosomes in yellow and arrowheads indicate some rough cER tubes in continuity with the CAB. (Insert) The cER network (red) and attached ribosomes (green) normally compacted in the CAB have been stretched away from the attached cortex under the force of shear. All acquisitions were made in Phallusia except B-D, which were made in Ciona.

View larger version (54K): [in a new window]   Fig. 6. Evolution of the cortex from fertilization to the eight-cell stage. Recapitulative diagram showing the rearrangements of the plasma membrane, adhering cER network and associated postplasmic/PEM RNAs (cER/mRNA), and microtubules from fertilization to the eight-cell stage. An oocyte, zygotes during the first and second major phases of reorganization, and an embryo at the eight-cell stage are represented in cross section and as isolated cortices in surface view and enlarged cross section.

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